Poly-β-hydroxyalkanoates (PHAs) are biological polyesters accumulated in cells by special microorganisms under special growth conditions.
The general formula of poly-β-hydroxyalkanoates is:

In which, n and m are integers from 1 to 4, and typically 1, that is for example 3-hydroxyalkanoates (3-HAS); R1 and R2 are straight chain or branched chain C1-12 alkyl groups which are substituted or non-substituted; X and Y are not 0 simultaneously, and determine the content of the component in copolymer. The average molecular weight of PHAs is generally 1-4 million Da.
The physical property of PHAs is similar to that of polypropylene. As its biodegradability, biocompatibility, piezoelectric properties and optical activity are characteristics not possessed by common petrochemical resins, it has wide application prospects in industry, agriculture, medicine, sanitation, food, electronics, etc.
Large scale industrialized production of PHAs has not been realized internationally. The principal reason is because the cost is much higher than that of petrochemical resin. The cost of PHAs includes mainly material cost and separation purification cost. The material cost depends on production efficiency of bacteria species and fermentation technology, whilst the separation purification cost depends largely on technology. The current extraction technology includes separation of cells from fermentation liquid with high speed centrifuge and purification of PHAs in separated wet bacterial body with organic solvent extraction, chemical reagent or surfactant+enzyme. These methods suffer from high cost or serious pollution, and are difficult to be industrialized. A one step extraction separation method for extracting polyhydroxyalkanoates directly from fermentation liquid containing cells is disclosed in Chinese patent application CN1328160A, but it must use large quantities of sodium hypochlorite and has the defect of poor operation environment, serious pollution, cost increased by waste water treatment, and product quality affected by shear degradation of PHAs.
Chinese patent No. CN119067A discloses a method for separating and purifying polyhydroxyalkanoates in bacterial cells from bacterial thallus. The method includes two steps in extraction, which requires separation of bacterial thallus from fermentation liquid. As the bacterial thallus is very small (several microns) and the fermentation liquid has certain viscosity, it is necessary to use high speed centrifuge of separation factor higher than 6000. This greatly increases the investment and is a bottleneck in large scale industrialized production. The method also requires expensive protease to treat the separated product which increases cost.
The extraction method disclosed in Chinese patent CN1171410A requires high speed centrifuge and lyophilization of product separated with centrifuge. These are very difficult in large scale industrialized production. Taking a factory producing 100,000 tons of PHAS/year as example, nearly 3000-4000 tons of fermentation liquid/day will be centrifuged and 600-800 tons will be lyophilized. The industrial cost will be very high. The final washing with organic solvent pollutes the environment and increases product cost.
U.S. Pat. No 4,910,145 discloses a method for separating and extracting PHAS with enzyme and surfactant. Since the cell wall and membrane are very complicated in composition and cannot be completely decomposed with one enzyme, it is necessary to use several enzymes or a compound enzyme. As the optimal action conditions as pH, temperature, etc. of different enzymes are different, the technology is very complicated. The method requires heating fermentation liquid to >80° C. which consumes enormous energy. The price of enzyme preparation is high, so the separation cost is high. Besides, the purity of product is not high.
The purpose of this invention is to provide an extraction method for PHAs, which can effectively reduce separation and purification cost, reduces pollution, and is suitable for industrialized production.
Invention
This invention provides a method for directly separating and purifying polyhydroxyalkanoates in cells from bacterial fermentation liquid, which comprises:    (1) pretreating fermentation liquid with a physical method for breaking cell wall;    (2) adjusting the pH of the pretreated fermentation liquid so that it is alkaline;    (3) adding anionic surfactant and agitating;    (4) separating and extracting coagulated precipitate in reaction liquid;    (5) washing and drying.
The physical method includes mechanical breaking and ultrasonic breaking. The sequence of adjusting pH and adding surfactant is interchangeable. The mechanical breaking includes ball milling or high pressure homogenization.
In step 3, aside from adding anionic surfactant, coagulating agent can be added.
The mechanical breaking method used to break cell wall can be ball milling.
The pH of the pretreated fermentation liquid is adjusted to 8-13. The alkaline substance used in adjusting pH can be solid or an aqueous solution of NaOH, Na2CO3, NaHCO3 or ammonia water.
The anionic surfactant can be olefinesulfonate (AOS), a fatty alcohol sulfate, a fatty alcohol polyoxyethylene-ether sulfate (AES), a fatty alcohol-polyoxyethylene ether (AEO), alkylphenol-polyoxyethylene ether, etc. The quantity of anionic surfactant is 0.5-20% (W/V) of fermentation liquid.
The coagulating agent is sodium polyacrylate, modified starch, polyamine, etc. The quantity of the coagulating agent is 0.5-20% (W/V) of the fermentation liquid.
After adding the anionic surfactant and the coagulating agent, the reaction temperature under agitation is 10-70° C. and the reaction time 5-60min.
Centrifuge, filter-press, vacuum suction filtration, etc. can be used for separating and extracting coagulated precipitate from the reaction liquid.
The invention is applicable to separation and purification of fermentation liquid of bacteria and its aberrance and gene engineering bacteria containing polyhydroxyalkanoates. Applicable bacteria include Alcaligenes, Pseudomonas, Azotobacter, Rhodospirillum, Methylotrophs, Bacillus, etc.
The invention has no high requirement for dry weight of cells and content of PHAs in fermentation liquid. The invention has the advantage of simple technology, low cost, high yield and greatly reduced pollution, so large scale industrialized production can be realized.